Ectopic germline recombination activity of the widely used Foxp3‐YFP‐Cre mouse: a case report

Summary Regulatory T (Treg) cell‐specific deletion of a gene of interest is a procedure widely used to study mechanisms controlling Treg development, homeostasis and function. Accordingly, several transgenic mouse lines have been generated that bear the Cre recombinase under control of the Foxp3 promoter either as a random transgene insertion or knocked into the endogenous Foxp3 locus, with the Foxp3YFP‐Cre strain of mice being one of the most widely used. In an attempt to generate Treg cells that lacked expression of the insulin receptor (Insr), we crossed Foxp3YFP‐Cre mice with Insrfl/fl mice. Using a conventional two‐band PCR genotyping method we found that offspring genotypes did not correspond to the expected Mendelian ratios. We therefore developed a quantitative PCR‐based genotyping method to investigate possible ectopic recombination outside the Treg lineage. With this method we found that ~50% of the F1‐generation mice showed evidence of ectopic recombination and that ~10% of the F2‐generation mice had germline Cre recombination activity leading to a high frequency of offspring with global Insr deletion. Use of the quantitative PCR genotyping method enabled accurate selection of mice without ectopic recombination and only the desired Treg cell‐specific Insr deletion. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3YFP‐Cre allele, particularly when studying genes that are systemically expressed. Foxp3‐YFP‐cre mice are commonly used to generate regulatory T (Treg) cell‐specific knockout mice but when crossed with insulin receptor floxed mice, we found that some offspring had recombination outside the Treg lineage. Ectopic recombination could not be identified using a conventional two‐band‐based PCR genotyping method so we developed a quantitative PCR method to discriminate between mice that had Treg cell‐specific versus ectopic recombination. Our data highlight the need to use genotyping methods that allow for assessment of possible ectopic recombination driven by the Foxp3‐YFP‐Cre allele.